A large fraction of the diseases in humans and animals that result from virus infections are caused by members of the picornavirus family. Like all picornaviruses, the encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) produce a protein called the 3C protease, which is absolutely required for virus replication in infected cells. The 3C proteases produced by EMCV and HAV, at least, are rapidly degraded by the cellular ubiquitin-proteasome system (UPS). The UPS, which is involved in the regulation of many critical cellular processes, operates by modifying proteins to be degraded with chains of the small protein ubiquitin in a process called polyubiquitylation, and the tagged proteins are degraded by the proteasome. The long-term objectives of the proposed project are to clarify the mechanisms by which the 3C proteases are marked for destruction and to discover the functions of the UPS-mediated destruction of the 3C proteases, as they relate to virus replication success. One specific aim of the project is to purify and identify the unknown ubiquitin- protein ligase, or ligases, that recognize the EMCV and HAV 3C proteases as ubiquitylation substrates and determine how the three ubiquitylation pathways known to target the 3C proteases function together to mark these viral proteins for degradation by the proteasome. The strategy to achieve this aim is to use protein purification and mass spectroscopy, along with interfering RNA library screening, to identify the unknown ligase, or ligases. The determination of how 3C protease polyubiquitylation occurs will be accomplished by measuring the kinetics with which the purified, reconstituted pathways catalyze the major phases of the polyubiquitylation process. A second aim is to determine the extent to which UPS-mediated 3C protease degradation in cells infected by EMCV affects virus replication success and the onset of cytopathic effects. The strategy to achieve this aim is to use interfering RNA to specifically inhibit the ubiquitylation pathways involved in 3C protease polyubiquitylation and measure the extent to which this inhibition alters new virion component production rates, infectious progeny virion yield, and the timing of cytopathicity onset. The proposed research is expected to provide a detailed characterization of the polyubiquitylation process by which the EMCV and HAV 3C proteases are tagged for degradation and to demonstrate the extent to which EMCV replication depends upon the UPS-mediated reduction of the viral 3C protease concentration. The findings will facilitate new research into the relationship between the UPS and other picornaviruses and may reveal new targets for antiviral therapies that disrupt the UPS. In addition, because little is known about how multiple protein-ubiquitylating pathways function together to catalyze protein polyubiquitylation, the findings from this research will likey be very useful in helping to explain how other proteins associated with human diseases are targeted and tagged for destruction. The project will support biomedical research infrastructure development because it includes a substantial undergraduate student research training component. PUBLIC HEALTH RELEVANCE: A large fraction of virus-associated diseases in humans is caused by infections with picornaviruses, but the mechanisms by which these viruses are replicated in their hosts are far from understood. The proposed project will provide important new insights into the links between picornavirus infection success and critical host cell components. This is likely in turn to result in the identification of new strategies for treating diseases caused by picornavirus infections.